Application of circPTK2 as marker in preparation of bladder cancer diagnosis or prognosis product

Abstract

The invention relates to the technical field of biological medicines, and relates to application of circPTK2 as a marker in preparation of a product for bladder cancer diagnosis or prognosis. Researches show that the CAFs-derived exosome can deliver circPTK2 to bladder cancer cells, the circPTK2 can be combined with C-Myc protein and promote nuclear translocation of the C-Myc protein, then expression of C-Myc downstream Warburg effect related proteins GLUT1 and LDHA is regulated and controlled, and migration, invasion and lymph node metastasis of the bladder cancer cells are affected. Meanwhile, the circPTK2 level in serum and urine exosomes is closely related to prognosis and lymphatic metastasis of bladder cancer patients and can be used as an effective marker for diagnosis and prognosis of bladder cancer and prediction of lymphatic metastasis, and a new thought and tool are provided for clinical diagnosis and treatment of bladder cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and specifically to the application of circPTK2 as a biomarker in the preparation of products for the diagnosis or prognosis of bladder cancer. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors worldwide and the leading cause of death among urinary system cancers. Based on the depth of tumor invasion, bladder cancer can be divided into non-muscle-invasive bladder cancer and muscle-invasive bladder cancer. Approximately 20%–30% of newly diagnosed patients have muscle-invasive bladder cancer, often accompanied by lymph node metastasis. Clinical studies show that the 5-year survival rate for patients with muscle-invasive bladder cancer without lymph node metastasis after radical cystectomy can reach over 70%. However, once lymph node metastasis occurs, even with postoperative adjuvant chemoradiotherapy, the 5-year overall survival rate is only 20%–30%. Therefore, lymph node metastasis is a key factor affecting the prognosis of bladder cancer patients.

[0003] Currently, there are no satisfactory treatment options for lymph node metastasis in bladder cancer, which remains a significant challenge in clinical practice. Traditional molecular targeted therapies often focus on molecular targets within bladder cancer cells themselves (such as VEGF, PI3K / AKT, and Nectin-4), but related clinical trials have not yielded satisfactory results, indicating that identifying therapeutic targets from within tumor cells has reached a bottleneck. In recent years, the role of the tumor microenvironment in tumor development has gradually gained attention. Cancer-associated fibroblasts (CAFs), as an important component of the tumor microenvironment, can regulate tumor cell growth, invasion, and metastasis through the secretion of cytokines, enzymes, or exosomes.

[0004] Therefore, identifying the key molecular mechanisms of lymph node metastasis in bladder cancer can provide a theoretical basis for finding precise markers and therapeutic targets, assist in the development of more effective molecularly targeted drugs, and has significant clinical and social benefits. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide the application of circPTK2 as a marker in the preparation of products for the diagnosis or prognosis of bladder cancer, thereby solving the problem of the lack of effective markers for lymph node metastasis in bladder cancer in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] This invention provides the application of circPTK2 as a biomarker in the preparation of bladder cancer diagnostic products.

[0008] In some embodiments of the present invention, the circPTK2 is derived from exosomes secreted by tumor-associated fibroblasts.

[0009] This invention provides the application of circPTK2 as a biomarker in the preparation of bladder cancer prognostic products.

[0010] In some embodiments of the present invention, the circPTK2 is derived from exosomes secreted by tumor-associated fibroblasts.

[0011] In some embodiments of the present invention, the diagnostic product is used to assist in determining whether a subject has bladder cancer.

[0012] In some embodiments of the present invention, the prognostic product is used to assist in assessing disease progression, lymph node metastasis risk, or survival prognosis in patients with bladder cancer.

[0013] In some embodiments of the present invention, the product includes reagents or kits for detecting the expression level of exosome circPTK2 in biological samples.

[0014] In some embodiments of the present invention, the biological sample is serum or urine.

[0015] In some embodiments of the present invention, the reagent includes primers for specifically amplifying circPTK2, wherein the forward and reverse primers of the primers cross the circumforming site of circPTK2.

[0016] In some embodiments of the present invention, the primers are used to detect the expression level of circPTK2 by real-time quantitative PCR.

[0017] The present invention also provides a kit for the diagnosis or prognosis of bladder cancer, comprising reagents for detecting the expression level of exosome circPTK2 in a biological sample, said biological sample being serum or urine, said reagents comprising primers for specifically amplifying circPTK2, said primers having a forward and reverse primer that cross the circumforming site of circPTK2.

[0018] In some embodiments of the present invention, at least one of exosome extraction reagent, RNA extraction reagent, reverse transcription reagent, and real-time quantitative PCR reaction reagent is included.

[0019] Based on the technical solution of this invention, compared with the prior art, this invention has the following beneficial effects: This invention has confirmed through research that CAF-derived exosomes can deliver circPTK2 to bladder cancer cells. circPTK2 can bind to C-Myc protein and promote its nuclear translocation, thereby regulating the expression of Warburg effect-related proteins such as downstream C-Myc proteins GLUT1 and LDHA, affecting the migration, invasion, and lymph node metastasis of bladder cancer cells. Simultaneously, the expression level of circPTK2 in serum and urinary exosomes is significantly correlated with the prognosis and lymph node metastasis of bladder cancer patients. Based on this, diagnostic or prognostic products prepared using circPTK2 as a biomarker can provide important evidence for the clinical diagnosis, prognostic assessment, and lymph node metastasis prediction of bladder cancer by detecting the expression level of circPTK2 in serum or urinary exosomes. These products have the advantages of simple operation, minimal invasiveness, high specificity and sensitivity, and help improve the early diagnosis rate and prognostic accuracy of bladder cancer, providing strong support for timely treatment and disease monitoring, and have significant clinical significance and application value. Attached Figure Description

[0020] Figure 1 This is a diagram showing the primary culture and identification results of CAFs; among them, Figure 1 In the figure, A represents the α-SMA immunofluorescence staining results of CAFs and normal fibroblasts (HFs); Figure 1 In the figure, B represents the Western blot result of CAFs-specific marker proteins.

[0021] Figure 2 Figure 1 shows the identification results of CAFs-derived exosomes; A is an electron micrograph of CAFs and HFs-derived exosomes, showing the typical cup-shaped morphology of exosomes; B is the expression of exosome-specific markers (such as CD9 and CD63) detected by immunoblotting, confirming that the extracted sample is an exosome; C is the exosome particle size analysis results, which are consistent with the particle size distribution characteristics of exosomes (30-150nm); D is the Transwell assay results, showing that CAFs-derived exosomes can significantly promote the migration and invasion of bladder cancer cells, while HFs-derived exosomes have no significant effect, clarifying the metastatic function of CAFs-derived exosomes.

[0022] Figure 3To elucidate the regulation of circRNA expression in bladder cancer cells by CAFs-derived exosomes. A and B represent high-throughput sequencing analysis of changes in circRNA expression profiles in bladder cancer cells after interaction with CAFs and HFs-derived exosomes, and intersection analysis to screen for differentially expressed circRNAs. C shows the qRT-PCR validation results, indicating that CAFs-derived exosomes significantly upregulated circPTK2 expression in bladder cancer cells, confirming that circPTK2 is a key circRNA delivered by CAFs-derived exosomes.

[0023] Figure 4 Exon analysis for circPTK2 origin.

[0024] Figure 5 The image shows the results of PCR performed using cDNA and gDNA as templates with circPTK2-specific primers and PTK2 linear primers, followed by gel electrophoresis of the PCR products.

[0025] Figure 6 After circPTK2 from CAF-derived exosomes was delivered to bladder cancer cells, the stability of the circ and linear RNA structures of PTK2 was observed using the actinomycin D assay.

[0026] Figure 7 The exonuclease assay confirmed that circPTK2 is resistant to exonuclease degradation, further supporting its stability.

[0027] Figure 8 RIP experiments were conducted to detect the enrichment of AGO2 on circFUT8 (positive control, whose binding to AGO2 has been confirmed in previously published articles) and circPTK2.

[0028] Figure 9 To perform CHIP experiments on C-Myc protein, primers for the GLUT1 and LDHA promoter regions were designed. PCR experiments showed that the GLUT1 and LDHA promoter sequences could be successfully amplified.

[0029] Figure 10 FISH experiments revealed that stable knockdown of circPTK2 in CAF-derived exosomes reduced the distribution of C-Myc in the nuclei of bladder cancer cells.

[0030] Figure 11 To stably knock down circPTK2 in CAF-derived exosomes and enable it to act on bladder cancer cells, we observed the mRNA level statistics of downstream genes of C-Myc.

[0031] Figure 12To understand the regulation of GLUT1 and LDHA proteins by C-Myc in protein imprinting experiments (left figure), and to understand the regulation of GLUT1 and LDHA proteins in bladder cancer cells by siRNA knockdown of circPTK2 in CAFs-derived exosomes.

[0032] Figure 13 This figure shows the effect of Warburg effector proteins GLUT1 / LDHA on bladder cancer migration and invasion.

[0033] Figure 14 The figure shows the results of in vivo experiments observing the effect of circPTK2 in CAF-derived exosomes on lymph node metastasis in bladder cancer. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0035] The following description is based on specific embodiments.

[0036] Example 1: Extraction and Identification of CAFs-Derived Exosomes

[0037] 1. Methods for isolating exosomes

[0038] (1) Primary culture and identification of CAFs

[0039] Bladder cancer tissue was minced, digested with 0.25% trypsin, filtered, and seeded into culture dishes. DMEM medium containing 10% fetal bovine serum was added, and the cells were cultured at 37°C in a 5% CO2 incubator. After cell adhesion and growth, CAFs were purified using a differential adhesion method. The expression of CAF-specific markers α-SMA and FAP was detected by immunofluorescence and Western blot to identify the obtained cells.

[0040] (2) Extraction and identification of CAFs-derived exosomes

[0041] The culture supernatants of CAFs and HFs were collected, and exosomes were extracted by ultracentrifugation (100,000 × g, 2 h). The morphology of exosomes was observed by transmission electron microscopy, the particle size distribution of exosomes was detected by nanoparticle tracking analysis (NTA), and the expression of exosome-specific markers CD63, CD9 and TSG101 was detected by Western blot to identify whether CAFs-derived exosomes were successfully extracted.

[0042] 3. Screening and Identification of circPTK2

[0043] CAF-derived exosomes and HF-derived exosomes were co-cultured with bladder cancer cells (T24, UM-UC-3), respectively. Differentially expressed circRNAs were screened using high-throughput sequencing. The expression level of circPTK2 was verified by qRT-PCR, and the circular structure of circPTK2 was verified by PCR and sequencing. The stability of circPTK2 was detected by actinomycin D assay and exonuclease assay.

[0044] 4. Research on the mechanism of circPTK2 regulating lymph node metastasis in bladder cancer

[0045] RIP experiment: RIP experiment was performed using C-Myc antibody, and qRT-PCR was used to detect the enrichment of circPTK2 in RNA precipitated by C-Myc antibody.

[0046] Dual-luciferase reporter gene assay: The circPTK2 truncated dual-luciferase reporter gene plasmid and the GLUT1 and LDHA promoter dual-luciferase reporter gene plasmid were constructed and co-transfected with the C-Myc overexpression plasmid into bladder cancer cells, and luciferase activity was detected.

[0047] Western blot and qRT-PCR experiments: circPTK2 in CAFs-derived exosomes was overexpressed or knocked down to detect the mRNA and protein expression levels of C-Myc, GLUT1, and LDHA in bladder cancer cells, with a focus on observing nuclear translocation changes of C-Myc.

[0048] Transwell assay: After knocking down circPTK2 in CAF-derived exosomes, they were co-cultured with bladder cancer cells to detect the migration and invasion abilities of bladder cancer cells.

[0049] 5. Experimental Results

[0050] (1) Results of primary culture and identification of CAFs

[0051] like Figure 1 As shown in Figure A, immunofluorescence revealed positive expression of α-SMA in CAFs; as Figure 1 As shown in Figure B, Western blot confirmed that the expression levels of α-SMA and FAP in CAFs were significantly higher than those in HFs, indicating that CAFs were successfully cultured and identified.

[0052] (2) Results of extraction and identification of CAFs-derived exosomes

[0053] like Figure 2As shown in A~C, transmission electron microscopy revealed that the exosomes were round or oval vesicles with a particle size distribution mainly concentrated in the range of 50-150 nm. Western blot confirmed that CD63, CD9 and TSG101 were positively expressed in the exosomes, indicating that CAFs-derived exosomes were successfully extracted.

[0054] (3) Screening and identification results of circPTK2

[0055] like Figure 3 A~C and Figures 4-7 As shown, high-throughput sequencing revealed that circPTK2 was significantly highly expressed in bladder cancer cells treated with CAFs-derived exosomes; PCR and sequencing confirmed that circPTK2 originated from the reverse splicing of exons 5-7 of the PTK2 gene; stability experiments showed that the half-life of circPTK2 exceeded 24 hours and it could withstand exonuclease degradation, confirming that circPTK2 is a stable circular RNA.

[0056] (4) Experimental results of the circPTK2 regulatory mechanism

[0057] like Figure 8 As shown in A and B, the RIP experiment showed that circPTK2 was significantly enriched in the RNA precipitated by C-Myc antibody, confirming that circPTK2 can bind to C-Myc protein.

[0058] like Figure 9 As shown in B, the dual-luciferase reporter gene assay showed that C-Myc can bind to specific regions of circPTK2 and specific sites of GLUT1 and LDHA promoters, promoting their transcriptional activity.

[0059] like Figures 10-12 As shown, Western blot and qRT-PCR experiments revealed that knocking down circPTK2 significantly reduced the nuclear translocation level of C-Myc, while also reducing the mRNA and protein expression levels of GLUT1 and LDHA.

[0060] like Figure 13 As shown in the Transwell assay, knocking down circPTK2 in CAF-derived exosomes significantly reduced the migration and invasion abilities of bladder cancer cells.

[0061] In summary, the experiment successfully completed the primary culture and identification of CAFs, clarifying the high expression characteristics of their specific markers α-SMA and FAP; it successfully extracted and identified CAF-derived exosomes, which exhibited typical morphology, particle size distribution, and specific markers; the screened circPTK2 was highly expressed in bladder cancer cells treated with CAF-derived exosomes and possessed a stable ring structure; the mechanistic study further confirmed that circPTK2 can bind to C-Myc protein, enhancing the migration and invasion ability of bladder cancer cells by regulating the nuclear translocation of C-Myc and the expression of downstream GLUT1 and LDHA, thereby promoting lymph node metastasis in bladder cancer. These results provide important evidence for a deeper understanding of the molecular mechanisms of lymph node metastasis in bladder cancer and lay a solid experimental foundation for the diagnosis and treatment of bladder cancer targeting circPTK2.

[0062] Example 2: shRNA targeting circPTK2 inhibits lymph node metastasis in an animal model.

[0063] 1. Construct a CAF cell line that can stably knock down circPTK2. The construction method includes: designing specific shRNA sequences targeting circPTK2 (shcircPTK2#1: 5'-GCTACAGTGGTTTGAAGAA-3'; shcircPTK2#2: 5'-GTGAAGAAGCTGCTGAAAT-3') and negative control shRNA (NC: 5'-TTCTCCGAACGTGTCACGT-3').

[0064] The synthesized shRNA sequence was cloned into a lentiviral vector (e.g., pLKO.1-puro), co-transfected with the packaging plasmid into 293T cells, and the lentiviral particles were collected and concentrated. CAF cells were then infected with the lentivirus. Forty-eight hours post-infection, puromycin (final concentration 2 μg / mL) was added for selection. Selection was continued for two weeks to obtain stably knocked-down CAF cell lines CAFs-Exo-shcircPTK2#1 and CAFs-Exo-shcircPTK2#2, and a negative control CAFs-Exo-NC cell line. The knockdown efficiency of circPTK2 was verified by qRT-PCR to ensure successful cell line construction.

[0065] 2. Exosome extraction: Exosomes (CAFs-Exo-shcircPTK2#1, CAFs-Exo-shcircPTK2#2 and CAFs-Exo-NC) were extracted from CAFs cell lines with knocked-down circPTK2. The extraction method was the same as described in Example 1, which was "ultra-high speed centrifugation (100000×g, 4℃ for 2h)". After extraction, the particle size distribution of the exosomes was 50-150nm.

[0066] (3) Tumor formation and intervention in nude mice:

[0067] UM-UC-3 cells in the logarithmic growth phase were taken and the concentration was adjusted to 1×10^7 cells / mL with PBS. 100 μL of cell suspension (containing 1×10^6 cells) was injected into the hind paw pad of each nude mouse.

[0068] Monitor tumor growth weekly after inoculation, until the tumor reaches a size of 50-100 mm. 3 At approximately 7-10 days old, the mice were randomly divided into 4 groups (n=5 / group):

[0069] PBS group: 100 μL of PBS was injected intratumorally;

[0070] CAFs-Exo-NC group: 100 μL of CAFs-Exo-NC (concentration of 200 μg / mL) was injected into the tumor.

[0071] CAFs-Exo-shcircPTK2#1 group: 100 μL of CAFs-Exo-shcircPTK2#1 (concentration of 200 μg / mL) was injected into the tumor.

[0072] CAFs-Exo-shcircPTK2#2 group: 100 μL of CAFs-Exo-shcircPTK2#2 (concentration of 200 μg / mL) was injected into the tumor.

[0073] One injection every 3 days, for a total of 4 injections.

[0074] (4) Lymph node metastasis assessment: Four weeks after intervention, the metastasis of popliteal lymph nodes was observed using the IVIS Lumina III in vivo imaging system (PerkinElmer): Nude mice were injected intraperitoneally with D-fluorescein (150mg / kg), and anesthetized and imaged 10 minutes later. The fluorescence intensity of the lymph node region (photons / second / square centimeter / sphericity) was quantified using Living Image software.

[0075] (5) Histological and molecular detection: After the experiment, the footpad tumor and popliteal lymph nodes were dissected, weighed, and their volume was measured. The calculation formula is: Volume = (Length × Width) / (Length × Width) 2 ) / 2.

[0076] Some tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned (4μm), stained with hematoxylin and eosin (HE), and observed under an optical microscope to determine the number and morphology of tumor metastases in the lymph nodes.

[0077] Another portion of the tissue was used for qRT-PCR detection: total RNA was extracted, reverse transcribed into cDNA, and the mRNA expression levels of circPTK2, C-Myc, GLUT1, and LDHA were detected;

[0078] Immunohistochemical analysis: After antigen retrieval, paraffin sections were incubated with primary antibodies (C-Myc: ab32072, Abcam; GLUT1: ab115730, Abcam; LDHA: ab47010, Abcam) and DAB staining was performed. The positive staining intensity (IOD / Area) was quantitatively analyzed using Image-ProPlus software to observe the nuclear localization of C-Myc (nuclear positive rate).

[0079] 3. Experimental Results

[0080] like Figure 14 As shown in the in vivo imaging results: compared with the PBS group and the CAFs-Exo-NC group, the fluorescence intensity of the popliteal lymph nodes in the CAFs-Exo-shcircPTK2#1 and #2 groups was significantly reduced (P<0.01), indicating a reduction in the number of lymph node metastases.

[0081] Histological results: HE staining showed that the area and number of metastatic tumor lesions in the popliteal lymph nodes of the CAFs-Exo-shcircPTK2 group were significantly less than those of the control group (P<0.01).

[0082] qRT-PCR showed that the mRNA expression levels of circPTK2, C-Myc, GLUT1, and LDHA in tumor and lymph node tissues of the CAFs-Exo-shcircPTK2 group were significantly lower than those in the control group (P<0.01).

[0083] Immunohistochemistry showed that the CAFs-Exo-shcircPTK2 group had a significantly lower C-Myc nuclear localization ratio (number of nuclear positive cells / total number of cells), and the protein expression intensity of GLUT1 and LDHA (IOD / Area) was significantly lower than that of the control group (P<0.01).

[0084] The above results are expected to confirm that shRNA targeting circPTK2 can effectively inhibit lymph node metastasis of bladder cancer in animal models by inhibiting the delivery of circPTK2 in CAF-derived exosomes and downregulating the C-Myc-GLUT1 / LDHA axis.

[0085] Example 3: Clinical Sample Analysis

[0086] 1. Experimental Materials

[0087] Serum and urine samples were collected from 200 bladder cancer patients and 50 healthy controls.

[0088] Inclusion criteria for bladder cancer patients: confirmed diagnosis of urothelial carcinoma of the bladder by pathological examination; no history of preoperative radiotherapy, chemotherapy, targeted therapy or immunotherapy; complete clinical data, including tumor stage, grade, lymph node metastasis status, etc.; age 18-80 years, and no serious underlying diseases of the heart, liver, kidneys or other organs.

[0089] Inclusion criteria for healthy controls: no history of cancer confirmed by physical examination, imaging examination and tumor marker testing; no urinary system diseases; age and sex matched with the bladder cancer patient group; and no significant underlying diseases.

[0090] 2. Experimental Methods

[0091] Sample processing: After serum sample collection, let stand at room temperature for 30 min, centrifuge at 3000×g for 10 min to separate serum, aliquot into RNase-free EP tubes, and store at -80℃; for urine sample collection, collect the first midstream urine of the morning, centrifuge at 3000×g for 15 min within 2 hours to remove impurities, aliquot the supernatant, and store at -80℃, avoiding repeated freeze-thaw cycles.

[0092] Exosome extraction: Exosomes were extracted from serum and urine using commercially available exosome extraction kits (such as Thermo Fisher Scientific’s Total Exosome Isolation Kit). The purity of the extraction was verified by Western blot detection of exosome-specific markers CD63, CD9 and TSG101. The particle size distribution (50-150 nm was considered acceptable) was detected by nanoparticle tracking analysis (NTA).

[0093] RNA extraction and detection: Total RNA was extracted from exosomes using RNase-free reagents. RNA purity was assessed using Nanodrop (A260 / A280 ratio 1.8-2.0), and RNA integrity was verified by agarose gel electrophoresis. cDNA was synthesized via reverse transcription and detected by qRT-PCR using specific primers designed for the circPTK2 circular junction. U6 snRNA was used as an internal control for serum exosomes, and GAPDH was used as an internal control for urinary exosomes. Three technical replicates were set up for each sample.

[0094] Data analysis: The relative expression level of circPTK2 was calculated using the 2^-ΔΔCt method, and its relationship with the clinicopathological parameters of patients was analyzed. Survival analysis was performed using the Kaplan-Meier method, and the differences in survival curves were compared using the Log-rank test.

[0095] 3. Experimental Results

[0096] The expression level of circPTK2 in serum and urinary exosomes of bladder cancer patients was significantly higher than that in healthy controls (P<0.01).

[0097] The expression level of circPTK2 in patients with positive lymph node metastasis was significantly higher than that in patients without lymph node metastasis (P<0.01).

[0098] Survival analysis showed that patients with high circPTK2 expression had significantly shorter disease-free survival and overall survival than those with low expression (P<0.01).

[0099] The above results confirm that circPTK2 can serve as a diagnostic marker and prognostic indicator for lymph node metastasis in bladder cancer.

[0100] Example 3: In vitro validation of circPTK2 as a therapeutic target

[0101] 1. Drug preparation

[0102] shRNA design: Specific shRNAs were designed targeting the circPTK2 sequence, namely sh1 (5'-GCTACAGTGGTTTGAAGAA-3') and sh2 (5'-GTGAAGAAGCTGCTGAAAT-3'), while the negative control shRNA (NC: 5'-TTCTCCGAACGTGTCACGT-3') sequence had no targeting.

[0103] Lentiviral packaging: sh1, sh2 and NC were cloned into the pLKO.1 lentiviral vector, and co-transfected into 293T cells with the packaging plasmids psPAX2 and pMD2.G. After 48-72 h of transfection, the supernatant was collected and the lentiviral particles were obtained by ultracentrifugation and concentration. The viral titer was measured (>1×10^8TU / mL).

[0104] Preparation of the injection: Lentiviral particles and liposomes (such as Lipofectamine 2000) are mixed at a volume ratio of 1:3 and incubated at room temperature for 15 min to prepare an injection containing lentivirus. It is stored at 4°C for later use and has a shelf life of 7 days.

[0105] 2. In vitro experiments

[0106] Cell culture: CAFs were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator until the logarithmic growth phase.

[0107] Drug treatment: CAFs were divided into 3 groups: blank control group (no treatment), NC group (infected with lentivirus containing NC), sh1 group (infected with lentivirus containing sh1), and sh2 group (infected with lentivirus containing sh2). The multiplicity of infection (MOI) was 10. The culture medium was changed 48 h after infection and cultured for another 72 h.

[0108] qRT-PCR detection: Culture supernatant of CAFs from each group was collected, exosomal RNA was extracted, and circPTK2 expression level was detected by qRT-PCR, the method being the same as in Example 6.

[0109] Transwell assay: CAF-derived exosomes were collected from each group and co-cultured with bladder cancer cells (T24, UM-UC-3) for 24 h. Cell migration ability was detected using Transwell chambers, and cell invasion ability was detected using Matrigel-coated Transwell chambers. Three replicates were set up for each group. After 24 h of culture, cells were fixed and stained, and the number of cells that penetrated the membrane was counted.

[0110] 3. Experimental Results

[0111] qRT-PCR analysis showed that, compared with the blank control group and the NC group, the expression level of circPTK2 in CAFs-derived exosomes in the sh1 and sh2 groups was significantly reduced (P<0.01).

[0112] Transwell assays showed that, compared with the blank control group and the NC group, the migration and invasion abilities of bladder cancer cells treated with CAFs-derived exosomes were significantly reduced in the sh1 and sh2 groups (P<0.01).

[0113] The above results confirm that the drug can effectively inhibit the expression of circPTK2, thereby inhibiting the migration and invasion of bladder cancer cells.

[0114] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0115] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of circPTK2 as a biomarker in the preparation of bladder cancer diagnostic products, characterized in that, The circPTK2 is derived from exosomes secreted by tumor-associated fibroblasts.

2. The application of circPTK2 as a biomarker in the preparation of bladder cancer prognostic products, characterized in that, The circPTK2 is derived from exosomes secreted by tumor-associated fibroblasts.

3. The application as described in claim 1, characterized in that, The diagnostic product is used to help determine whether a subject has bladder cancer.

4. The application as described in claim 2, characterized in that, The prognostic product is used to help assess disease progression, lymph node metastasis risk, or survival prognosis in patients with bladder cancer.

5. The application according to claim 1 or 2, characterized in that, The product includes reagents or kits for detecting the expression level of exosome circPTK2 in biological samples.

6. The application according to claim 5, characterized in that, The biological sample is serum or urine.

7. The application according to claim 5, characterized in that, The reagent includes primers for specifically amplifying circPTK2, wherein the forward and reverse primers of the primers cross the circPTK2 circumforming site.

8. The application according to claim 7, characterized in that, The primers were used to detect the expression level of circPTK2 by real-time quantitative PCR.

9. A kit for the diagnosis or prognosis of bladder cancer, characterized in that, The reagent comprises a reagent for detecting the expression level of exosome circPTK2 in a biological sample, said biological sample being serum or urine, said reagent comprising primers for specifically amplifying circPTK2, said primers having a forward and reverse primer that cross the circumforming site of circPTK2.

10. The reagent kit according to claim 9, characterized in that, It also includes at least one of the following: exosome extraction reagent, RNA extraction reagent, reverse transcription reagent, and real-time quantitative PCR reaction reagent.

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